A recombinant Escherichia coli for preparing soluble EC-SOD and its application

Abstract

The present disclosure provides a recombinant Escherichia coli for preparing soluble EC-SOD and its application, relating to the field of bioengineering technology. In the present disclosure, the genes of SOD and KGF2 are ligated using a coding sequence of a linker peptide containing a protease cleavage site and cloned into a pET28a expression vector. The pET28a expression vector is transformed into competent Escherichia coli BL21(DE3) cells to construct the recombinant Escherichia coli. The obtained recombinant Escherichia coli can solve the problem that EC-SOD forms inactive inclusion bodies during expression in Escherichia coli. It is applicable to the preparation of soluble EC-SOD.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a recombinant Escherichia coli for preparing soluble EC-SOD and its application. Background Art

[0002] Human extracellular superoxide dismutase (EC-SOD) is one of the three known SOD (superoxide dismutase) isozymes, and is a secreted glycoprotein containing Cu and Zn metal ions. EC-SOD mainly exists in the extracellular matrix and on the cell surface, and is the main SOD isozyme in extracellular fluids such as plasma, lymph fluid, synovial fluid and cerebrospinal fluid. It is also the main enzyme for scavenging extracellular O 2- , and has significant effects on reducing blood sugar, blood lipids and blood pressure. EC-SOD has extremely low content in tissues and is difficult to isolate and purify.

[0003] The Escherichia coli expression system has many advantages such as clear genetic background, high expression level, fast reproduction, low cost, good stability, strong anti-pollution ability and easy purification of products. However, when EC-SOD is expressed in the Escherichia coli expression system, the expression products are all inactive inclusion bodies. In addition, since EC-SOD is a four-subunit metalloenzyme and each subunit contains 6 sulfhydryl groups, the refolding rate of inclusion bodies is relatively low, the denaturation and refolding operation time is long, and the recovery efficiency of active proteins is low.

[0004] Fusion protein expression is an important protein expression strategy to enhance the solubility of target proteins. The coding sequence of the target protein is fused with the coding sequence of other proteins, and the target protein is synthesized by expressing the gene of the fusion protein. Human keratinocyte growth factor 2 (KGF2), also known as fibroblast growth factor 10 (FGF10), consists of 208 amino acids and has acid and heat instability. KGF2 has a wide range of biological functions, can promote epidermal hyperplasia and development, and has wide applications in the medical and cosmetic fields. There is currently no report on using KGF2 as a fusion tag to promote the soluble expression of heterologous proteins in Escherichia coli. Summary of the Invention

[0005] The purpose of the present invention is to improve the soluble expression level of EC-SOD in Escherichia coli by constructing recombinant Escherichia coli.

[0006] On the one hand, the present invention provides a recombinant Escherichia coli for preparing soluble EC-SOD.

[0007] The recombinant Escherichia coli is prepared by inserting the gene encoding KGF2 downstream of the gene encoding SOD in an expression vector, connecting the genes of SOD and KGF2 with a linker peptide coding sequence, and then transforming the SOD-KGF2 fusion protein expression vector into host Escherichia coli competent cells.

[0008] Further, the host Escherichia coli is Escherichia coli BL21(DE3).

[0009] Further, the expression vector is the expression vector pET28a.

[0010] An embodiment of the present invention provides a recombinant Escherichia coli, which can achieve soluble expression of EC-SOD. The recombinant Escherichia coli is obtained by connecting the SOD gene (removing the stop codon TAA) of the sequence shown in SEQ ID NO.1 and the KGF2 gene containing the sequence shown in SEQ ID NO.2 in an expression vector with a linker peptide coding sequence containing a protease cleavage site, and then transforming it into host Escherichia coli competent cells.

[0011] Further, the linker peptide containing a protease cleavage site has a TEV protease cleavage site. Further, the linker peptide containing a protease cleavage site has a nucleic acid sequence shown in SEQ ID NO.11 and an enzyme cleavage site sequence shown in SEQ ID NO.12.

[0012] On the other hand, the present invention provides a method for constructing the above recombinant Escherichia coli for soluble expression of EC-SOD, including the following steps:

[0013] (1) Synthesize the SOD nucleotide sequence according to the codon preference of Escherichia coli from the amino acid sequence of superoxide dismutase SOD, and clone it into the expression vector PET28a to construct the vector pET28a-SOD. Among them, the SOD nucleotide sequence is shown in SEQ ID NO.1.

[0014] (2) Synthesize the KGF2 nucleotide sequence according to the codon preference of Escherichia coli from the amino acid sequence of keratinocyte growth factor 2, and clone it into the expression vector pUC19 to construct the vector pUC19-KGF2. Among them, the KGF2 nucleotide sequence is as shown in SEQ ID NO.2.

[0015] (3) Using the vector pET28a-SOD as a template, amplify the pET28a-SOD vector backbone with primers P1 (SEQ ID NO.3) and primers P2 (SEQ ID NO.4) to obtain fragment 1.

[0016] (4) Using the vector pUC19-KGF2 as a template, the KGF2 fragment with a linker peptide was amplified using primer P3 (SEQ ID NO.5) and primer P4 (SEQ ID NO.6), denoted as fragment 2. This linker peptide is a linker peptide containing a protease cleavage site. This linker peptide has a TEV (tobacco etch virus) protease cleavage site. Further, this linker peptide has the nucleotide sequence shown in SEQ ID NO.11 and the cleavage site sequence shown in SEQ ID NO.12.

[0017] (5) Fragment 1 and fragment 2 were incubated using a one-step seamless cloning kit (ClonExpress II One Step Cloning Kit) to obtain a fusion protein expression vector.

[0018] (6) The fusion protein expression vector obtained in step (5) was used to transform Escherichia coli BL21(DE3) competent cells to obtain recombinant Escherichia coli E. coli BL21(DE3) / pET28a-SOD-KGF2.

[0019] On the other hand, the present invention provides an application of a recombinant Escherichia coli for soluble expression of EC-SOD in the preparation of EC-SOD.

[0020] On another aspect, the present invention provides a method for preparing EC-SOD using Escherichia coli. The recombinant Escherichia coli is cultured, an inducer is added to induce the expression of a soluble SOD-KGF2 fusion protein, and then the SOD-KGF2 fusion protein is proteolytically cleaved to obtain soluble EC-SOD and soluble KGF2. Compared with the prior art in which SOD protein is expressed in the form of inactive inclusion bodies, the present invention realizes the soluble expression of SOD. This method can obtain the SOD-KGF2 fusion protein in a soluble expression manner, and then obtain soluble EC-SOD by proteolytic cleavage, while retaining the activity of EC-SOD. At the same time, soluble KGF2 can also be obtained.

[0021] Further, the Escherichia coli is the recombinant Escherichia coli for preparing soluble expression of EC-SOD as described above E. coli BL21(DE3) / pET28a-SOD-KGF2. The recombinant Escherichia coli was inoculated into an LB liquid medium to obtain a seed solution; the obtained seed solution was inoculated into an LB liquid medium and cultured until the OD600 of the bacteria reached 0.6 - 0.8, and an inducer was added to induce the expression of the SOD-KGF2 fusion protein; the expressed fusion protein was proteolytically cleaved to obtain EC-SOD and KGF2.

[0022] Furthermore, the inducer can be IPTG (isopropyl-β-D-thiogalactoside), eliminating the need to use methanol as an inducer, which is highly safe and has little harm. In the method of the present invention, the inducer can be added at a final concentration of 0.05 - 0.5 mM to induce the expression of the recombinant Escherichia coli.

[0023] Furthermore, the protease is TEV (tobacco etch virus) protease. In the method of the present invention, 200 U of TEV protease is added to every 1 mg of the fusion protein and incubated overnight at 4°C.

[0024] The technical solution of the present invention has the following advantages:

[0025] The present invention induces the expression of the SOD-KGF2 fusion protein in Escherichia coli, realizes the soluble expression of EC-SOD in Escherichia coli, and simultaneously obtains two protein products, soluble SOD and KGF2, after protease cleavage, reducing the production cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0027] Figure 1 It is the electrophoresis diagram of the SOD protein: Lane 1 is Marker; Lane 2 is the precipitate of the cell lysate induced at 37°C; Lane 3 is the supernatant of the cell lysate induced at 37°C; Lane 4 is the precipitate of the cell lysate induced at 28°C; Lane 5 is the supernatant of the cell lysate induced at 28°C;

[0028] Figure 2 It is the vector map of pET28a-SOD-KGF2;

[0029] Figure 3 It is the electrophoresis diagram of the SOD-KGF2 fusion protein: Lane 1 is Marker; Lane 2 is the supernatant of the cell lysate induced at 37°C; Lane 3 is the precipitate of the cell lysate induced at 37°C; Lane 4 is the supernatant of the cell lysate induced at 28°C; Lane 5 is the precipitate of the cell lysate induced at 28°C; Lane 6 is the supernatant of the cell lysate of the control cells without plasmid induced at 28°C;

[0030] Figure 4 It is the electrophoresis diagram of the SOD protein and KGF2 after lysis and purification. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0032] Example 1 E. coli Construction and expression of BL21(DE3) / pET28a-SOD

[0033] According to the codon preference of Escherichia coli, the nucleotide sequence (SEQ ID NO.1) was synthesized from the amino acid sequence of superoxide dismutase SOD, cloned into the expression vector pET28a to obtain the vector pET28a-SOD. The vector pET28a-SOD was transformed into the competent cells of Escherichia coli BL21(DE3) strain, and positive transformants were selected. Colony PCR verification was performed using primer SOD-F (SEQ ID NO.7) and primer SOD-R (SEQ ID NO.8), denoted as strain E.coli BL21(DE3) / pET28a-SOD, and stored at -80 °C.

[0034] The above-constructed engineering strain E.coli BL21(DE3) / pET28a-SOD was cultured in LB containing 100 μg / mL Kan (kanamycin kanamycin ) Luria-Bertani)Streak on a solid plate and place it upside down in a constant temperature incubator at 37 °C for 12 - 16 h. Pick a single colony and inoculate it into LB liquid medium containing 100 μg / mL Kan, and culture it for 12 h at 37 °C and 200 rpm to obtain a seed solution. Then transfer the seed solution to fresh LB liquid medium containing 100 μg / mL Kan at an inoculation amount of 2% (v / v), and culture the bacteria until the OD600 reaches 0.6 - 0.8 at 37 °C and 200 rpm. Add IPTG (Isopropyl-beta-D-thiogalactopyranoside) with a final concentration of 0.2 mM, induce expression at 37 °C and 28 °C, 200 rpm for 12 h, then centrifuge at 4 °C and 8000 rpm for 10 min, discard the supernatant, collect the wet bacterial cell precipitate, resuspend it with 10 volumes of 100 mM PBS buffer with a pH of 7.0 (weigh 17.03 g of Na2HPO4 and 10.89 g of KH2PO4 and dissolve them in 100 ml of deionized water), break it with an ultrasonic cell disruptor, collect the cell lysate, centrifuge at 4 °C and 8000 rpm for 10 min, and take the supernatant and precipitate respectively for SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) verification. The 12% SDS-PAGE result of EC-SOD is as Figure 1 shown. The results show that after IPTG induction, EC-SOD mainly exists in the precipitate of ultrasonically disrupted bacterial cells and exists in the form of inclusion bodies.

[0035] Example 2 Construction and Expression of an Engineering Bacterium Expressing the Fusion Protein SOD-KGF2

[0036] According to the codon preference of Escherichia coli, synthesize the KGF2 nucleotide sequence (SEQ ID NO.2) from the amino acid sequence of keratinocyte growth factor 2, and ligate it into the cloning vector pUC19 to construct the pUC19-KGF2 vector.

[0037] Using pET28a-SOD as a template, the vector backbone of pET28a-SOD (denoted as fragment 1) was amplified using the upstream primer P1 (SEQ ID NO.3) and the downstream primer P2 (SEQ ID NO.4). Using pUC19-KGF2 as a template, the KGF2 fragment with a linker peptide (denoted as fragment 2) was amplified using the upstream primer P3 (SEQ ID NO.5) containing the linker peptide coding sequence and the downstream primer P4 (SEQ ID NO.6). There is a 20-base homologous arm between fragment 1 and fragment 2. Fragments 1 and 2 were incubated using the ClonExpress II One Step Cloning Kit (Nanjing Novoprotein Scientific Co., Ltd.) so that the KGF2 fragment with a linker peptide was inserted before the stop codon of SOD, and the expression vector as shown in Figure 2 was constructed. Then it was transformed into Escherichia coli BL21(DE3) competent cells. Positive transformants were selected and verified by colony PCR using the primer SOD-KGF2-F (SEQ ID NO.9) and the primer SOD-KGF2-R (SEQ ID NO.10). The constructed fusion protein expression vector was denoted as pET28a-SOD-KGF2 ( Figure 2 ), and the obtained strain was denoted as E.coli BL21(DE3) / pET28a-SOD-KGF2, which was stored at -80 °C.

[0038] The induction expression method of E.coli BL21(DE3) / pET28a-SOD-KGF2 was the same as that in Example 1. The 12% SDS-PAGE results of the SOD-KGF2 fusion protein are respectively as shown in Figure 3 . After induction with IPTG, the SDS-PAGE protein electrophoresis analysis results of the SOD-KGF2 fusion protein showed that the molecular weight was around 44 KD, which was consistent with the molecular weight of the target protein. The SOD-KGF2 fusion protein mainly existed in the supernatant of the sonicated bacteria, indicating that the fusion protein was mainly expressed in a soluble form. The nucleotide sequences of each primer and the target protein are shown in Table 1 in detail.

[0039] Table 1 Nucleotide sequences of each primer and the target protein

[0040]

[0041] Example 3 Cleavage of the SOD-KGF2 fusion protein and purification of EC-SOD and KGF2 proteins

[0042] (1) Cleavage of the fusion protein

[0043] Collect the E. coli BL21(DE3) / pET28a-SOD-KGF2 cells induced for expression by IPTG in Example 2, suspend them in 20 mM Tris-HCl buffer (pH 7.5) containing 0.3 M NaCl and 10 mM EDTA-2Na, and sonicate them in an ice bath for 15 minutes using an ultrasonic cell disruptor, with each sonication lasting 1 s and an interval of 2 s. Add recombinant TEV (tobacco etch virus) protease to the cell supernatant for lysis (the TEV protease was purchased from YEASON). For every 1 mg of the fusion protein, add one-tenth volume of 10x TEV protease buffer (500 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1% Tween-20 (v / v), 10 mM DTT) and 200 U of TEV protease, and incubate overnight at 4°C. The fusion protein is completely cleaved to obtain a cell lysate containing the EC-SOD protein with carboxyl-terminal residual Glu-Asn-Leu-Tyr-Phe-Gln amino acid residues and the KGF2 protein with a Gly residue remaining at the amino terminus. Centrifuge the obtained cell lysate at 8000 r / min for 10 min at 4°C, take the supernatant, and place it on ice for later use.

[0044] (2) Purification of human EC-SOD

[0045] Take the supernatant obtained in step (1) and use a UNOsphere S strong cation exchange chromatography column (40×12.6 mm, Biorad). The equilibration buffer and elution buffer A are 6 M urea and 0.25 M acetic acid, pH 4.0; the elution buffer B is 6 M urea, 0.25 M acetic acid, and 1 M sodium chloride, pH 4.05. The elution gradient starts from 100% elution buffer A for 5 minutes, then gradually goes to 70% elution buffer A / 30% elution buffer B for 10 min, and with a linear gradient of 30 - 100% elution buffer B for 50 min. Elute the column at a flow rate of 1 mL / min and collect the EC-SOD eluent in 10 mL tubes. After elution, wash the column with 100% elution buffer B for 20 min and equilibrate it with 100% equilibration buffer for 20 min. Monitor the elution at 280 nm to obtain the purified SOD protein solution. The electrophoresis pattern of the purified SOD protein is as Figure 4 shown.

[0046] (3) Purification of KGF2 protein

[0047] Use a CM-Sepharose 6FF ion exchange column and rinse the ion exchange column at a flow rate of 3.0 mL / min for 3 - 5 column volumes to remove the 20% ethanol solution in the column. Place pump A into a 20 mM phosphate buffer solution (pH 6.0) containing 0.3 M NaCl and 10 mM EDTA-2Na to balance the ion exchange column at a flow rate of 3.0 mL / min. After the UV display line becomes horizontal, end the rinsing. Adjust the flow rate to 1.0 mL / min and load the crude KGF2 sample. At a flow rate of 3.0 mL / min, pump A and pump B are respectively placed with degassed ddH2O and a 20 mM phosphate buffer solution (pH 6.5) containing 1.0 M NaCl and 10 mM EDTA-2Na for gradient elution of approximately 3 - 5 column volumes, and collect the elution peak, which is the purified KGF2 protein solution, and store it at -20°C. The electrophoresis pattern of the purified KGF2 protein is as shown in Figure 4 shown.

[0048] Example 4 Determination of EC-SOD Enzyme Activity

[0049] Perform enzyme activity assays on the EC-SOD obtained by IPTG-induced expression in Example 1, the SOD-KGF2 fusion protein obtained by IPTG-induced expression in Example 2, and the EC-SOD purified in Example 3, respectively.

[0050] For the enzyme activity assay, add the buffer according to Table 2 below, incubate in a 25°C constant temperature water bath for 20 min, add pyrogallol, mix well quickly with a shaker, place it in a 1 cm cuvette, and use the blank as a control. Measure the absorbance A every 0.5 min at a wavelength of 325 nm for 3 min continuously, and control the autoxidation rate A0 at 0.060 (± 0.001) / min. For the SOD activity assay, add samples according to Table 2 below, record the A1 value in the same way, and control the pyrogallol oxidation rate at 50% of the autoxidation rate, that is, 0.030 (± 0.001) / min, by adjusting the concentration of the SOD sample solution.

[0051] Table 2

[0052]

[0053] The definition of an enzyme activity unit is that the amount of enzyme that inhibits the autoxidation rate of pyrogallol by 50% per minute in 1 mL of the reaction solution is defined as one enzyme activity unit.

[0054] The calculation formula for enzyme activity units:

[0055] SOD activity (U) = (((A0 - A1) / A0) / 50%) * (Va / V1) * (N / VE)

[0056] A0——Autoxidation rate; A1——Oxidation rate after adding enzyme; N——Dilution factor; V1——Defined volume; Va——Total reaction volume; VE——Volume of enzyme added.

[0057] 1 mg of enzyme protein was taken respectively to determine the enzyme activity, that is, the specific enzyme activity of EC-SOD obtained in Example 1, the fusion protein obtained in Example 2, and the EC-SOD obtained by separation and purification in Example 3 was measured. The measurement results are shown in Table 3.

[0058] Table 3: Measurement results of EC-SOD specific enzyme activity

[0059]

[0060] The results further show that EC-SOD is expressed as inactive inclusion bodies in the engineering strain E. coli BL21(DE3) / pET28a-SOD. Therefore, under the induction condition of 28 °C, the specific enzyme activity of EC-SOD in the supernatant after cell disruption and centrifugation is extremely low. The fusion protein is mainly expressed in a soluble form in the engineering bacterium E. coli BL21(DE3) / pET28a-SOD-KGF2. It mainly exists in the supernatant of the cells after ultrasonic disruption. Therefore, under the induction condition of 28 °C, a relatively high EC-SOD enzyme activity was measured in the supernatant of the cells after ultrasonic disruption. The purified EC-SOD showed a maximum specific enzyme activity of 1803.8 U / mg.

[0061] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0062] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention without departing from the principles and purposes of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

[0063] As described above, it is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A recombinant Escherichia coli for preparing soluble EC-SOD, characterized in that, The recombinant Escherichia coli is obtained by removing the 3'-terminal stop codon TAA from the SOD gene of the sequence shown in SEQ ID NO.1 in an expression vector, ligating it with the KGF2 gene of the sequence shown in SEQ ID NO.2 using a linker peptide coding sequence containing a protease cleavage site, and then transforming it into a host Escherichia coli competent cell; The nucleotide sequence of the linker peptide containing the protease cleavage site is as shown in SEQ ID NO.11, and the amino acid sequence is as shown in SEQ ID NO.

12.

2. The recombinant Escherichia coli for preparing soluble EC-SOD according to claim 1, characterized in that, The host Escherichia coli is Escherichia coli BL21(DE3).

3. The recombinant Escherichia coli for preparing soluble EC-SOD according to any one of claims 1 to 2, characterized in that, The expression vector is the expression vector pET28a.

4. A method for constructing a recombinant Escherichia coli for preparing soluble EC-SOD according to any one of claims 1 to 2, characterized in that, It includes the following steps: (1) Synthesize the SOD nucleotide sequence according to the Escherichia coli codon preference from the superoxide dismutase amino acid sequence, and clone it into the expression vector PET28a to construct the pET28a-SOD vector; (2) Synthesize the KGF2 nucleotide sequence according to the Escherichia coli codon preference from the keratinocyte growth factor 2 amino acid sequence, and ligate it into the cloning vector pUC19 to construct the pUC19-KGF2 vector; (3) Using the pET28a-SOD vector as a template, amplify the pET28a-SOD vector backbone with primer P1 and primer P2, denoted as fragment 1; (4) Using the pUC19-KGF2 vector as a template, amplify the KGF2 fragment with the linker peptide coding sequence using primer P3 and primer P4 containing the linker peptide coding sequence, denoted as fragment 2; (5) Incubate fragment 1 and fragment 2 using a one-step seamless cloning kit, and then transform Escherichia coli BL21(DE3) competent cells to obtain the recombinant Escherichia coli E.coli BL21(DE3) / pET28a-SOD-KGF2; The sequence of primer P1 is as shown in SEQ ID NO.3, and the sequence of primer P2 is as shown in SEQ ID NO.4; the sequence of primer P3 in step (4) is as shown in SEQ ID NO.5, and the sequence of primer P4 is as shown in SEQ ID NO.

6.

5. The construction method of the recombinant Escherichia coli for preparing soluble EC-SOD according to claim 4, characterized in that, There is a 20-base homologous arm between fragment 1 and fragment 2 in step (5).

6. Use of the recombinant Escherichia coli according to any one of claims 1 to 2 in the preparation of EC-SOD.

7. The application according to claim 6, characterized in that Cultivate the recombinant Escherichia coli and add an inducer for induced expression, and the cell lysate is protease-digested to obtain two proteins, EC-SOD and KGF2.

Images